One of the principal mechanisms by which cellular regulation is effected is through the transduction of extracellular signals across the membrane that in turn modulate biochemical pathways within the cell. Protein phosphorylation represents one course by which intracellular signals are propagated from molecule to molecule resulting finally in a cellular response. These signal transduction cascades are highly regulated and often overlapping as evidenced by the existence of many protein kinases as well as phosphatases. It is currently believed that a number of disease states and/or disorders are a result of either aberrant expression or functional mutations in the molecular components of kinase cascades. Consequently, considerable attention has been devoted to the characterization of these proteins.
Nearly all cell surface receptors use one or more of the MAP kinase cascades during signal transduction. One subgroup of the MAP kinases is the MAPK/ERK kinases or MEKs. There are five members of the MEK family, two of which, MEK1 and MEK2, lie directly downstream of the Raf oncogene pathway. However, several other upstream components have been shown to activate MEKs including kinases in the c-Mos, and MEKK pathways (Robinson and Cobb, Curr. Opin. Cell Biol., 1997, 9, 180-186). MEKs catalyze both serine/threonine and tyrosine phosphorylation of their target molecules and do so in an ordered fashion (Seger et al., J. Biol. Chem., 1992, 267, 14373-14381).
MEK1 (also known as MEK1a, MAPKK, MKK1a, MAP kinase kinase and MAP kinase/ERK kinase) is a member of the MEK family of dual-specificity kinases and represents a convergent target for the regulation of a diverse set of cellular processes including proliferation, differentiation and development. MEK1 was first identified as an ERK activator in growth factor treated cells and was later cloned and characterized as a mitogen-activated protein kinase of ERK (Zheng and Guan, J. Biol. Chem., 1993, 268, 11435-11439). MEK1 is expressed in very low levels in most embryonic murine tissues but can be detected in skeletal muscle. It is expressed at much higher levels in adult tissue, particularly the brain. From these studies, it has been suggested that MEK1 is the MEK responsible for the proliferative or mitogenic response of tissues (Brott et al., Cell Growth Differ., 1993, 4, 921-929). The downstream targets of MEK1 identified to date are ERK1 and ERK2. Subsequent to phosphorylation, ERKs activate nuclear, membrane, cytosolic and cytoskeletal targets that in turn mediate multiple signaling cascades (Seger and Krebs, The FASEB Journal, 1995, 9, 726-735). Therefore, manifestations of altered MEK1 regulation can appear in many downstream events, the most widely investigated being the development of cancer.
Cellular transformation and acquisition of the metastatic phenotype are the two main changes normal cells undergo during the progression to cancer. Studies of dominant-negative and constitutive-active forms of MEK1 showed that sustained activation of MEK1 is necessary and sufficient for PC12 differentiation, transformation of NIH3T3 cells, and for the HRG-induced differentiation of a human breast carcinoma cell line (Cowley et al., Cell, 1994, 77, 841-852; Lessor et al., J. Cell Biochem., 1998, 70, 587-595). In addition, MEK1 was shown to be overexpressed (52% of individuals studied) in renal cell carcinomas (Oka et al., Cancer Res., 1995, 55, 4182-4187) and in human hepatocellular carcinoma (Schmidt et al., Biochem Biophys Res Commun, 1997, 236, 54-58). Other studies have demonstrated that oncogenes capable of transforming mammary gland epithelium require specific signal transduction pathways and that mammary tumors initiated by neu, v-Ha-ras, and c-myc have high levels of MEKs. Furthermore, the anchorage independent growth of these tumors was inhibited by the MEK specific inhibitor, PD 098059 (Amundadottir and Leder, Oncogene, 1998, 16, 737-746).
To date, strategies aimed at inhibiting MEK1 function have involved the use of anti-MAPKK antibodies, the chemical inhibitor PD 098059, antisense vector technology and dominant-negative forms of MEK1.
The most widely used inhibitor of MEK1 is the chemical moiety, PD 098059. This compound has been used extensively to demonstrate the involvement of MEKs in signaling cascades. It was shown to act as a noncompetitive inhibitor of MEKs by blocking activation by Raf and MEK kinases (Alessi et al., J Biol Chem, 1995, 270, 27489-27494). However, it has recently been proposed that this highly selective inhibitor of MEK1 competes with apoptotic signals thereby producing cells that avoid apoptosis and contribute to pathologic conditions (Mohr et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 5045-5050).
Studies designed to investigate the effect of bufalin on c-Jun N-terminal protein kinase (JNK) and on the signaling pathways downstream of MEK1 used vector-based antisense targeted to MEK1 in addition to a constitutively active mutant of MEK1. The activation of JNK, the induction of apoptosis, and the transcriptional activity of AP-1 which is transiently enhanced by the treatment with bufalin were all suppressed by the expression of the entire 1.3 kb MEK1 cDNA in antisense orientation. In addition, expression of a constitutively active mutant form of MEK1 induced the activation of AP-1 and subsequent apoptosis in U937 cells (Watabe et al., Oncogene, 1998, 16, 779-787).
In studies of Xenopus oocyte maturation, microinjection of anti-MAPKK neutralizing antibodies prevented the mos-induced metaphase arrest of the cell cycle, implicating MEK in cell cycle control (Kosako et al., J Biol Chem, 1994, 269, 28354-28358).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of MEK1. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting MEK1 function.
Antisense oligonucleotides, therefore, provide a promising new pharmaceutical tool for the effective modification of the expression of specific genes.